Our investigations have demonstrated that ribosomal protein S1 from E. coli (and an analogous protein from Caulobacter crescentus) is in many respects analogous to DNA helix-unwinding (single stranded nucleic acid-binding) proteins in that it stoichiometrically disrupts the secondary structure of many helical and stacked single stranded synthetic and natural polyribo- and polydeoxyribonucleotides and converts them into their fully or partially denaturated forms. S1 is involved in the binding of natural mRNA to the ribosomes and its presence on 30S subunits is indispensable for the formation of a 30S initiation complex with natural mRNA. 30S subunits deficient in S1 retain activity with AUG as mRNA and are also active in the poly U-directed binding of Phe-tRNA. A mono-N-ethylmaleimide derivative of S1 is nearly devoid of any RNA helix-unwinding properties but it readily incorporated into 30S subunits deficient in S1. The resulting NEM-S1 containing 30S subunits are completely inactive in the binding of labeled coliphage MS2 RNA and in the formation of an initiation complex with MS2 RNA as mRNA. They retain activity in the binding of the initiator fMet-tRNA in response to the trinucleotide AUG an in the binding of Phe-tRNA in response to poly U. These results suggest that a correlation exists between the RNA helix-unwinding capacity of isolated S1 and the function of S1 in the ribosomal binding of natural mRNA when the protein becomes part of the 30S subunit. Preliminary results indicate that a rabbit reticulocyte protein required for the binding of natural mRNA to ribosomes has RNA unwinding properties analogous to the properties of S1.